Viruses have developed many unique strategies to evade the host immune response in the pursuit of a common goal: to proliferate. In fact, most viruses use multiple tactics simultaneously to achieve this goal. Such is the case for poxviruses which have at least three mechanisms to prevent their hosts from detecting dsRNA, thereby preventing the activation of host innate immune sensors. One of these mechanisms is to use viral encoded decapping enzymes D9 and D10 to clear accumulating dsRNA by removing the protective 5 cap of host and viral mRNAs, committing them to degradation by cellular 5-3 exoribonuclease Xrn1. The fact that D9 and D10 are expressed at different stages of the viral replication cycle and early studies indicate they recognize capped mRNA differently suggests D9 and D10 have distinct functions, perhaps targeting different mRNAs during infection. However, despite the significant role these enzymes play in host immune evasion and the extensive body of literature describing poxvirus pathogenesis, we lack an effective molecular understanding of how they both recognize their capped mRNA and catalyze cap hydrolysis to evade the host immune response. This research plan seeks to combine biochemistry, structural biology and virology to establish the molecular basis with which these enzymes recognize and hydrolyze their substrates, and how their substrate specificity contributes to poxvirus pathogenesis. To determine if substrate specificity is conferred during substrate binding or the catalytic step, in vitro binding and activity assays will be performed using various substrates relevant to the different mRNAs present during poxvirus infection. The molecular determinants that govern substrate recognition will be identified using high- and low-resolution structural techniques. The combination of high- and low-resolution techniques will build a more complete understanding of the specific molecular interactions, conformational changes, and higher order assembly that contribute to function. Mutational analyses using in vitro binding and activity assays in addition to cell-based infectivity assays will be used to link structure to phenotype and to validate the biochemical and biological relevance of the structural model. Lastly, protein-protein interaction partners will be identified using affinity purification coupled with mass spectrometry to determine if substrates are selected by enzyme-substrate binding per se or if protein cofactors assist in recruiting D9 and D10 to target mRNAs. Together, these studies will provide a molecular understanding of how substrate recognition and protein-protein interactions with poxvirus decapping enzymes control target mRNA selection and cap cleavage during infection. Understanding poxvirus decapping enzyme activity at the molecular level is an important step toward a comprehensive model of mRNA stability during poxvirus infection that can be used in developing poxvirus tools for use in immunotherapy as well as to create novel antiviral therapeutics as a defense against future threats of epidemics and bioterrorism.